Immunostaining of Drosophila polytene chromosomes to investigate recruitment of chromatin-binding proteins

Methods Mol Biol. 2012;809:267-77. doi: 10.1007/978-1-61779-376-9_18.

Abstract

Gene transcription is a complex process that involves a large number of proteins. These proteins can be brought to their target genes by a variety of different mechanisms: many transcription factors interact with specific DNA sequences in promoters or enhancers, several epigenetic regulators bind histones bearing specific modifications, elongation factors and some RNA processing factors bind to the transcribing RNA polymerase, and other factors interact directly with nascent transcripts or noncoding RNA. Immunostaining of Drosophila polytene chromosomes allows the genome-wide localization of factors involved at different stages of transcriptional regulation. In this chapter, we present protocols that adapt the general technique to probe different recruitment mechanisms employed by these factors, including specific interactions with phosphorylated RNA polymerase II and RNA-mediated chromatin associations.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chromosomal Proteins, Non-Histone / genetics
  • Chromosomal Proteins, Non-Histone / metabolism
  • Drosophila
  • Drosophila Proteins / genetics
  • Drosophila Proteins / metabolism*
  • Heat-Shock Proteins / genetics
  • Heat-Shock Proteins / metabolism
  • Polytene Chromosomes / genetics
  • Polytene Chromosomes / metabolism*
  • Protein Binding
  • RNA Polymerase II / genetics
  • RNA Polymerase II / metabolism
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Transcriptional Elongation Factors / genetics
  • Transcriptional Elongation Factors / metabolism

Substances

  • Chromosomal Proteins, Non-Histone
  • Drosophila Proteins
  • Heat-Shock Proteins
  • Transcription Factors
  • Transcriptional Elongation Factors
  • SPT5 transcriptional elongation factor
  • RNA Polymerase II